Faster, cheaper DNA sequencing method developed

December 20, 2009

A team of researchers led by Boston University biomedical engineer Amit Meller is using electrical fields to efficiently draw long strands of DNA through nanopore sensors, drastically reducing the number of DNA copies required for a high throughput analysis. Figure copyright, Nature Nanotechnology, 2009

(PhysOrg.com) -- Boston University biomedical engineers have devised a method for making future genome sequencing faster and cheaper by dramatically reducing the amount of DNA required, thus eliminating the expensive, time-consuming and error-prone step of DNA amplification.

In a study published in the Dec. 20 online edition of Nature Nanotechnology, a team led by Boston University Biomedical Engineering Associate Professor Amit Meller details pioneering work in detecting DNA molecules as they pass through silicon nanopores. The technique uses electrical fields to feed long strands of DNA through four-nanometer-wide pores, much like threading a needle. The method uses sensitive electrical current measurements to detect single DNA molecules as they pass through the nanopores.

"The current study shows that we can detect a much smaller amount of DNA sample than previously reported," said Meller. "When people start to implement genome sequencing or genome profiling using nanopores, they could use our nanopore capture approach to greatly reduce the number of copies used in those measurements."

Currently, genome sequencing utilizes DNA amplification to make billions of molecular copies in order to produce a sample large enough to be analyzed. In addition to the time and cost DNA amplification entails, some of the molecules - like photocopies of photocopies - come out less than perfect. Meller and his colleagues at BU, New York University and Bar-Ilan University in Israel have harnessed electrical fields surrounding the mouths of the nanopores to attract long, negatively charged strands of DNA and slide them through the nanopore where the DNA sequence can be detected. Since the DNA is drawn to the nanopores from a distance, far fewer copies of the molecule are needed.

Before creating this new method, the team had to develop an understanding of electro-physics at the nanoscale, where the rules that govern the larger world don't necessarily apply. They made a counterintuitive discovery: the longer the DNA strand, the more quickly it found the pore opening.

"That's really surprising," Meller said. "You'd expect that if you have a longer 'spaghetti,' then finding the end would be much harder. At the same time this discovery means that the nanopore system is optimized for the detection of long DNA strands -- tens of thousands basepairs, or even more. This could dramatically speed future genomic sequencing by allowing analysis of a long DNA strand in one swipe, rather than having to assemble results from many short snippets.

"DNA amplification technologies limit DNA molecule length to under a thousand basepairs," Meller added. "Because our method avoids amplification, it not only reduces the cost, time and error rate of DNA replication techniques, but also enables the analysis of very long strands of DNA, much longer than current limitations."

With this knowledge in hand, Meller and his team set out to optimize the effect. They used salt gradients to alter the electrical field around the pores, which increased the rate at which DNA molecules were captured and shortened the lag time between molecules, thus reducing the quantity of DNA needed for accurate measurements. Rather than floating around until they happened upon a nanopore, DNA strands were funneled into the openings.

By boosting capture rates by a few orders of magnitude, and reducing the volume of the sample chamber the researchers reduced the number of DNA molecules required by a factor of 10,000 - from about 1 billion sample molecules to 100,000.

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It looks like they only optimized conditions for DNA capture by the pore, ..

I believe you've misread the report:

"The technique uses electrical fields to feed long strands of DNA through four-nanometer-wide pores, much like threading a needle. The method uses sensitive electrical current measurements to detect single DNA molecules as they pass through the nanopores."

So, individual bases seem to be identified by changes in an electrical current.

No, the part you quoted is about using the current to detect when the molecule enters and exits the pore. I've been following this line of research for quite some time, the idea of using the nanopore for sequencing is 20 years old and there is just one major challenge - identifying the bases as they pass through the pore. If they managed to do it it would be clearly stated in the article.

I was on a seminar about this method a year ago - in numerical simulations identifying bases is already very difficulthttp://pubs.acs.o...l0601076so what about real situations, which are no so idealized with repeatable nanoelectrodes ...

Maybe we could somehow use nature's ability to read/work with DNA - somehow mount ribosome or polymerase and somehow monitor its state ... For example connect it electrically using antibodies specific to some two its part and 'cable' made of nanotubes or even of (-CH=CH-). Resistance should somehow depend on 'processed' base ...And so with the structure of resonances - maybe some optical/magneto-optical method?

Ok - I think I might have a practical idea - get this information from the speed of the process.We can literally observe polymerase processing DNA - to process succeeding base it has to get from the environment the proper nucleoside triphosphate - there are only four of them - we can manipulate their concentrations.If we would choose different concentrations for them, there would be correlations between type of the base and time of its processing - by watching such many processes we could determine the sequence.

There's one aspect of this that baffles me. So you use a positively charged pore to attract a DNA molecule to it. You may even be able to get the DNA molecule to "thread" itself through the pore, to a degree. However, how on earth do you keep the DNA molecule continuously moving through the pore, and eventually exiting the pore? After all, it is attracted to the pore, and once there, it won't want to leave!

The pore itself is not charged, two charged electrodes are located in ionic solution on both sides of the pore and the resulting electric fields draws DNA through the pore and towards positively charged electrode.

Yes, but pyrosequencing uses separate steps for each nucleotide - each of them requires macroscopic time...If we want sequence whole human genome in a few days at cost of a few thousand dollars, there are required completely different approaches - generally there are two ways: massive parallelization or using methods which require microscopic time for one base - like making that it goes through a nanopore/protein/ribosome and somehow identify bases by the way. There is also approach with mounting ssDNA to surface and use atomic force microscope.

If someone is interested, there has started discussion about this sequencing watching speed of polymerase while large concentration differences of 'nucleotide carriers' (like 1:10:100:1000)http://www.thesci...p=225199

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